Hydroxycholesterol immunoassay

ABSTRACT

Provided is a derivative of 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. Also provided is a protein conjugated to the above derivative. Further provided is an antibody composition comprising antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. Additionally, a method of making antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Also, a method of assaying for 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Additionally provided is a kit for detecting 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. A method of detecting an enzyme or enzymes utilized in phase II drug metabolism is also provided. Also, a method of detecting an enzyme that synthesizes 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Further provided is a method of evaluating progression of multiple sclerosis in a patient. Also provided is a method of determining whether a treatment for multiple sclerosis in a patient is effective. Further, a method of evaluating progression of Huntington&#39;s disease in a patient is provided. Additionally provided is a method of determining whether a treatment for Huntington&#39;s disease in a patient is effective.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present application generally relates to antibody detection ofmetabolites. More specifically, methods and compositions are providedfor preparing and using antibodies to detect and quantify24S-hydroxycholesterol.

(2) Description of the Related Art

24S-hydroxycholesterol (24HC) (structure below) is one of severalmono-oxygenated metabolites of cholesterol.

Excess cholesterol in the brain is converted to 24HC by cholesterol24-hydroxylase (CYP46). Owing to its ability to cross the blood-brainbarrier, 24HC is present in peripheral circulation. Because CYP46 ispredominantly expressed in the brain, 24HC in plasma originates almostexclusively from the brain (Lütjohann et al., 1996). Interestingly,certain CYP46 single nucleotide polymorphisms are associated withAlzheimer's disease (“AD”)(Garcia et al., 2009). Plasma levels of 24HCcorrelate with brain and hippocampal size (Bretillon et al., 2000;Koschack et al., 2009). 24HC in plasma or cerebrospinal fluid is anindicator of brain cholesterol turnover, particularly from myelin, anddemyelination caused by neuronal degeneration results in an increasedflux of 24HC across the blood-brain barrier and into plasma. Thus,active neuronal cells are the major source of 24HC in human circulationand the concentration of 24HC in plasma is considered a surrogate markerfor brain cholesterol homeostasis. Additionally, 24HC is a signalingmolecule in the brain, inducing ApoE-mediated cholesterol efflux fromastrocytes by a direct effect on ApoE transcription, protein synthesisand secretion (Leoni et al., 2010). Direct toxic and inflammatory geneexpression-inducing effects of 24HC may also be involved in thepathology of AD or other cognitive dysfunctions (Alexandrov et al.,2005; Misch et al., 2004; Zhao et al., 2009). Further, neurodegenerationand the resulting loss of neurons has been found in some studies toresult in the reduction of 24HC in plasma in AD, Huntington's disease,and multiple sclerosis (Björichem et al., 2009; Irizarry, 2004; Leoni,2009; Leoni et al., 2008; Masterman et al., 2002; Solomon et al., 2009;Teunissen et al., 2003, 2005; Valenza et al., 2007). However, otherstudies have found increased 24HC plasma levels in AD patients(Lütjohann and von Bergmann, 2003; Lütjohann et al., 2000). Incerebrospinal fluid, 24S-hydroxycholesterol levels increase, and appearto reflect the rate of neurodegeneration (Leoni et al., 2010;Papassotiropoulos et al., 2002; Shafaati et al., 2007). Reduced plasmalevels of 24HC are also induced by statin treatment (Lütjohann and vonBergmann, 2003; Vega and Weiner, 2007). In a further association withneurodegenerative disease, gene therapy with CYP46 in a mouse model ofAD, administered before the onset of amyloid deposits, reduced Aβpeptides, amyloid deposits and trimeric oligomers, and improved spatialmemory in those mice (Hudry et al., 2010). Additionally, ablation ofacyl-CoA:cholesterol acetyltransferase 1 (ACAT1) in the brain leads to areduction of AD-associated forms of amyloid precursor protein andincreases in brain 24HC (Bryleva et al., 2010).

There is a close relationship between the biotransformation of drugs andnormal biochemical processes occurring in the human body. The metabolismof drugs involves many pathways associated with the synthesis ofendogenous substrates such as steroid hormones, cholesterol and bileacids. It should be recognized that many of the enzymes involved in drugmetabolism are principally designed for the metabolism of theseendogenous compounds. These enzymes metabolize drugs only because thedrugs resemble the natural substrate.

During phase II metabolism, a substrate is rendered more hydrophilicthrough the covalent attachment of an endogenous molecule. The cytosolicsulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT) familiesof enzymes account for the majority of phase II metabolism in humans andanimals. Sulfonation and glucuronidation are generally consideredcompeting pathways. Typically, sulfonation predominates at low substrateconcentrations, while glucuronidation predominates at higherconcentrations.

In both brain and liver, 24HC serves as a liver-X receptor (TAR) agonistand has an important role in cholesterol homeostasis. Conversion of thismetabolite to bile acids accounts for only 40-50% of the metabolite'selimination, leaving a large percentage of 24HC metabolism and excretionoccurring by other pathways. The SULT and UGT enzymes represent a highlyresponsive defense system against the mutagenicity of carcinogenicenvironmental chemicals and the toxicity of xenobiotics and endogenousmetabolic intermediates. Conjugation with either sulfonate or glucuronicacid has been implicated as important for biliary excretion of 24HC.

Cytosolic SULTs are involved with the conjugation of therapeutic drugs,xenobiotics and small endogenous compounds including hydroxysteroids,thyroid hormones, estrogens, bile acids, cholesterol and oxysterols.24HC can be conjugated by at least three isoforms of human cytosolicSULTs, but others (e.g. SULTs 1A1, 1 A3, 1B1, 1C1) display nodiscernable activity (Cook et al., 2009). SULTs 2A1 and 1E1 sulfonateboth the 3- and 24-hydroxyl groups to form24-hydroxycholesterol-3,24-disulfate. SULT2B1b forms only24-hydroxycholesterol-3-sulfate. The 3-sulfate as a monosulfate or asthe disulfate can be hydrolyzed by human placental steroid sulfatase,whereas the 24-sulfate is resistant to its actions. Both the24-hydroxycholesterol-3-sulfate and 24-sulfate are antagonists of LXRactivation.

Current methods for determination of 24HC generally involve gaschromatography-mass spectrometry, or liquid chromatography-tandem massspectrometry (see, e.g., Berkard et al., 2004; DeBarber et al., 2008;Leoni et al., 2008). Because the importance of 24HC in human physiologyand disease is being increasingly recognized, and because the currentmethods for identifying 24HC are time consuming and require expensiveequipment, there is a need for a simpler assay for 24HC. The presentinvention addresses that need.

BRIEF SUMMARY OF THE INVENTION

The inventors have developed methods for preparing antibodies to24S-hydroxycholesterol (24HC). The prepared antibodies have lowcross-reactivity to related metabolites, and are useful reagents forspecific and sensitive immunoassays for 24HC.

Thus, in some embodiments, a derivative of 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol is provided. The derivative comprises a moietycomprising a free amino, carboxylic acid, or sulfhydryl group covalentlyattached to the 3-OH position of the hydroxycholesterol.

Also provided is a protein conjugated to the above derivative.

Additionally, a composition is provided comprising the above proteincombined with an adjuvant that can improve an antibody immune responseto the protein in a vertebrate injected with the composition.

Further provided is an antibody composition comprising antibodies thatspecifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol.

A method of making the above-described antibodies is also provided. Themethod comprises: (a) preparing an immunogen by conjugating theabove-described derivative to a carrier protein; (b) immuMzing avertebrate with the imrnunogen under conditions such that the immunesystem of the vertebrate makes the antibodies; and (c) taking theantibodies from the vertebrate.

Additionally provided is a method of assaying for 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol in a fluid or tissue sample from a mammal. Themethod comprises combining the sample with the above-describedantibodies, then determining whether the antibodies specifically bind to22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol from the sample. In thesemethods, specific antibody binding to hydroxycholesterol from the sampleindicates that the hydroxycholesterol is present in the sample.

A kit for detecting and/or quantifying 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol is further provided. The kit comprises theabove-described antibodies.

The present invention is also directed to a method of detecting anenzyme or enzymes utilized in phase II drug metabolism in a sample,where the enzyme or enzymes are capable of altering22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol. The method comprisescombining the sample with 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterolfor a time sufficient for the enzyme or enzymes to modify the22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol, then assaying the sampleusing the above immunoassay, wherein a reduction in the amount of the22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol detected by the assayindicates that the sample comprises the enzyme or enzymes.

Additionally provided is a method of detecting an enzyme thatsynthesizes 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholest rol ina sample. The method comprises combining the sample with cholesterol fora time sufficient for the enzyme to modify the cholesterol, thenassaying the sample using the method of claim 43, wherein an increase inthe amount of 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesteroldetected by the assay indicates that the sample comprises the enzyme.

Also provided is a method of evaluating progression of multiplesclerosis in a patient. The method comprises measuring24S-hydroxycholesterol in blood plasma or serum of the patient over timeby the above-described assay method. In this method, progression ofmultiple sclerosis is indicated by decreasing levels of24S-hydroxycholesterol in the blood plasma or serum over time.

A method of determining whether a treatment for multiple sclerosis in apatient is effective is also provided. The method comprises measuring24S-hydroxycholesterol in blood plasma or serum of the patient over timeby the above-described assay method. In this method, the treatment iseffective if the 24S-hydroxycholesterol levels do not decrease overtime.

Further provided is a method of evaluating progression of Huntington'sdisease in a patient. The method comprises measuring24S-hydroxycholesterol in blood plasma or serum of the patient over timeby the above-described assay method. In this method, progression ofHuntington's disease is indicated by decreasing levels of24S-hydroxycholesterol in the blood plasma or serum over time.

A method of determining whether a treatment for Huntington's disease ina patient is effective is additionally provided. The method comprisesmeasuring 24S-hydroxycholesterol in blood plasma or serum of the patientover time by the above-described assay method. In this method, thetreatment is effective if the 24S-hydroxycholesterol levels do notdecrease over time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a standard curve of 24S-hydroxycholesterol in an ELISAusing an antiserum elicited against3-O-succinoyl-24S-hydroxycholesterol.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

The inventors have prepared antibodies to 24S-hydroxycholesterol (24HC)that have low cross-reactivity to related metabolites. The antibodiesare useful reagents in rapid, specific and sensitive immunoassays for24HC that can be utilized with standard immunoassay protocols. Based onthese results, the inventors believe that effective antibodies with lowcross-reactivity could also be made to other hydroxycholesterolmetabolites, for example 22-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol. Thus, the inventorsenvision that any of the methods relating to 24HC that are discussedherein could also be utilized with 22-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol.As such, while most of the discussion below addresses only compositionsand methods relating to 24HC for the sole purpose of simplifying thereadability of this application, the inventors believe that any of thecompositions and methods could be equally applied to22-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol and27-hydroxycholesterol without undue experimentation. The inventorstherefore do not disclaim, and specifically include,22-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol and27-hydroxycholesterol as envisioned for any of the compositions andmethods below that discuss 24HC.

Since 24HC is a hapten that is too small to illicit an antibody immuneresponse by itself, it must be covalently coupled to a carrier protein.The 24HC-protein conjugate, when injected into a vertebrate, presents24HC to the vertebrate's immune system as an epitope on the protein,such that antibodies are generated to the 24HC epitope. However, 24HCdoes not have a moiety to which it could be conveniently conjugated to acarrier protein. Therefore, a derivative of 24HC is preferably preparedthat has a reactive moiety for conjugation to proteins or otherreagents.

Antibodies against the closely related compound cholesterol have beenprepared (Yinsong et al., 2007; Biró et al., 2007; Sato et al., 1976;Dijkstra et al., 1996). However, antibodies made by conjugation of acarrier protein to the cholesterol 3-OH moiety showed significantlydiminished cholesterol binding activity when compared to antibodies madeby injection of cholesterol-rich liposomes containing the adjuvantmonophosphoryl lipid A (Dijkstra et al., 1996). Thus, the excellentresults achieved herein in producing effective antisera against 24HC, asdescribed in the Example below, were surprising and unexpected.

Any derivative providing a reactive moiety that can be conjugated to aprotein can be utilized herein. As is known in the art, moietiescomprising a free amino group, a free carboxylic acid group, or a freesulfhydryl group provide useful reactive groups for protein conjugation(See, e.g., the website atpiercenet.com/browse.cfm?fldlD4E018AA6-5056-8A76-4E57-3BC84C88A328). Forexample, a free amino group can be conjugated to proteins viaglutaraldehyde cross-linking, or via carbodiimide cross-linking toavailable carboxy moieties on the protein. Also, a hapten with a freesulfhydryl group can be conjugated to proteins via maleimide activationof the protein, e.g., usingsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC), then linkage to the sulfhydryl group.

Because the 24-hydroxyl group is the feature that distinguishes 24HCfrom cholesterol and other naturally occurring hydroxycholesterols(e.g., 22-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol, 27-hydroxycholesterol), the inventors reasonedthat, in spite of the poor results achieved with cholesterol in similarschemes, conjugation of the carrier protein to the 3-OH moiety wouldpresent the branched hydroxyalkane end of the 24HC molecule as anepitope of the carrier molecule to the immune system, increasing thelikelihood that antibodies elicited to the 24HC would have lowcross-reactivity with cholesterol and other hydroxycholesterols.

Thus, in some embodiments, a derivative of 24S-hydroxycholesterol isprovided. The derivative comprises a moiety comprising a free amino,carboxylic acid, or sulfhydryl group covalently attached to the 3-OH.

In some embodiments, the moiety is a carboxylic acid. A carboxylic acidmoiety can be conjugated to the 3-OH of 24HC by any method known in theart, including any method resulting in the following linkages:

In some embodiments, the derivative is conjugated through an esterlinkage, for example via the conjugation of a dicarboxylic acid to the24HC. Any dicarboxylic acid can be utilized, here. Nonlimiting examplesinclude oxalic acid, malonic acid, succinic acid, glutaric acid, adipicacid, pimelic acid, suberic acid, azelaic acid, sebacic acid,undecanedioic acid, dodecanedioic acid, and a phthalic acid. Methods forconjugating dicarboxylic acid derivatives to hydroxyl moieties are knownin the art. See, e.g., Example. In some embodiments, the derivativeconsists of 3-O-succinoyl-24S-hydroxycholesterol:

In other embodiments, the derivative comprises3-O-succinoyl-24S-hydroxycholesterol. Such derivatives can furtherencompass any other useful moiety, e.g., a moiety that further assistsin conjugation to a protein. An example of the latter is anN-hydroxysuccinimide ester moiety. Thus, in some embodiments, the3-O-succinoyl-24S-hydroxycholesterol further comprises anN-hydroxysuccinimide ester conjugated to the carboxyl group on thesuccinoyl moiety. In some of these embodiments, the compound is

Another useful group that can be conjugated to the3-O-succinoyl-24S-hydroxycholesterol is a moiety that can be utilizedfor signaling. Nonlimiting examples of signaling moieties includechromophores, fluorophores, and luminescent moieties. Derivatizedcholesterol comprising fluorophores are known in the art. See, e.g.,Sparrow et al. (1999) and Jennings et al. (1999). Any fluorophore can beutilized in these compositions. Additionally, such fluorescenthydroxycholesterols can utilize hydroxycholesterol derivatives otherthan the 3-O-succinoyl derivative. For example, a hydroxycholesterolester-BODIPY dye, analogous to the cholesterol ester-BODIPY dyedescribed in Jennings et al., 1999 can be utilized. Further, besides thevarious positions along the ring structure of 24S-hydroxycholesterolother than the 3β-hydroxyl position can be derivatized with a variety oftags to enable the described assay formats, so long as this attachmentdoes not interfere with the binding of the described antibodies to the24-OH moiety. As an example, N,N-dimethyl amino naphthalene sulfonate(dansyl) hydroxycholesterol can be utilized. Such a compound isanalogous to the dansyl-cholesterol described in McIntosh et al. (2008).Compare with the compounds described in U.S. Patent ApplicationPublication US 2008/0177059, describing BODIPY-cholesterol derivativeswhere the BODIPY dye is conjugated to the 22 position of cholesterol. Ananalogous derivative of a hydroxycholesterol would not bind to theanti-hydroxycholesterol antibodies described herein, since theantibodies are designed to bind where the BODIPY dye would be conjugatedin such compounds.

Another exemplary group for signaling purposes is biotin. Thus, in someembodiments, the compound further comprises a biotin conjugated to thecarboxyl group on the succinoyl moiety. Such a compound can alsousefully comprise a linkage between the carboxy group, e.g., on thesuccinoyl moiety, and the biotin, to provide a space between the twogroups, preventing steric hindrance of; e.g., a streptavidin-enzymeconjugate that binds to the biotin. The spacer can be of any length orconstruction known in the art. In some such embodiments, the compound is

Also provided herewith is a protein conjugated to any of the abovederivatives, for example the derivative3-O-succinoyl-24S-hydroxycholesterol. The protein can be conjugated tothe derivative using any of methods known in the art, for example asdiscussed above, e.g., glutaraldehyde, a maleimide reagent, acarbodiimide, or an activated ester, e.g., an N-hydroxysuccinimideester. The protein can be any protein that can usefully be utilized withthe derivative. For example, the protein can be an enzyme, e.g., as asignaling molecule in enzyme immunoassays. Nonlimiting examples includehorseradish peroxidase and alkaline phosphatase. The protein can also bea fluorescent protein, e.g. for the same purpose.

In other embodiments, the protein is a carrier protein for immunizationof a vertebrate, to make antibodies against the derivative. Many suchproteins are known in the art, e.g., keyhole limpet hemocyanin, bovineserum albumin, ovalbumin, etc. In some embodiments, a composition isprovided comprising the above protein-derivative conjugate combined withan adjuvant that can improve an antibody immune response to the proteinin a vertebrate injected with the composition. In certain specificembodiments the derivative is 3-O-succinoyl-24S-hydroxycholesterol.

Further provided herewith is an antibody composition comprisingantibodies that specifically bind to 22-hydroxycholesterol, 24HC,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol.In some embodiments, the antibodies specifically bind to 24HC (“24HCantibodies”). In some of these embodiments, the 24HC antibodies haveless than 1% cross-reactivity to cholesterol, 22-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol, 27-hydroxycholesterol andDHEA when compared to the binding of the antibodies to 24HC. In otherembodiments, the antibodies have less than 0.2% cross-reactivity to22-hydroxycholesterol, cholesterol and DHEA when compared to the bindingof the antibodies to 24S-hydroxycholesterol. In additional embodiments,the antibodies have less than 0.01% cross-reactivity to cholesterol whencompared to the binding of the antibodies to 24S-hydroxycholesterol.

These antibodies can be immunoglobulins of any vertebrate species, e.g.,rabbit, goat, mouse, sheep, chicken, etc. and can be polyclonal ormonoclonal. They can include the Fc region or they can be Fab or Fab2fragments. Additionally, they can be from any source, e.g., from theserum of an animal injected with an immunogen such as any of theimmunogens described above, or they can be from culture or ascites as isknown in the art of hybridoma technology. Alternatively, they can befrom recombinant sources, e.g., as described in Winter et al., 1994, orCharlton and Porter, 2002.

A method of making 24HC antibodies is also provided. The methodcomprises: (a) preparing a 24HC immunogen by conjugating any of theabove-described 24HC derivatives to a carrier protein; (b) immunizing avertebrate with the immunogen under conditions such that the immunesystem of the vertebrate makes antibodies to 24HC; and (c) taking the24HC antibodies from the vertebrate. In some embodiments, the derivativeis 3-O-succinoyl-24S-hydroxycholesterol. The carrier protein can be anyof those discussed above. In some of these embodiments, the carrierprotein is keyhole limpet hemocyanin, bovine serum albumin (BSA) orovalbumin.

These methods are not narrowly limited to any particular set ofvertebrates. In some embodiments, the vertebrate is a rabbit, a goat, amouse, a chicken, or a sheep. Additionally, the antibodies taken fromthe vertebrate can be polyclonal antibodies or monoclonal antibodies.

The 24HC antibodies described above can be utilized to detect 24HC byany immunoassay known in the art. Thus, additionally provided herein isa method of assaying for 24HC in a fluid or tissue sample from a mammal.The method comprises combining the sample with 24HC antibodies, thendetermining whether the antibodies specifically bind to 24HC from thesample. In these methods, specific antibody binding to 24HC from thesample indicates that 24HC is present in the sample.

The sample can be from any fluid or tissue from the mammal. In someembodiments, the sample is from cerebrospinal fluid, blood (e.g., wholeblood, serum or plasma), or brain tissue, since those samples are knownto contain 24HC.

The sample can be from any mammal. In some embodiments, the sample isfrom a human, e.g., a human without any apparent disease, or a humanthat has, or is suspected of having, cognitive impairment, Huntington'sdisease, Alzheimer's disease, or multiple sclerosis, which are known toaffect the amount of 24HC in cerebrospinal fluid, plasma, and/or braintissue.

Depending on the assay and sample, the sample can be used directly inthe assay, for example after dilution in a buffer, or the sample can beat least partly purified to eliminate substances that might interferewith the assay. Numerous such purification procedures are known in theart. For example, the sample could be solvent-extracted or subjected tochromatography, e.g., through a C18 column, before use in the assay. Insome protocols, the eluate from the chromatographic procedure orextraction is dried and resuspended in a buffer that does not interferein the assay. See, e.g., Axelson (1991). Such a drying and resuspensionprocedure can also be used to concentrate the sample if there is concernthat the amount of the 24HC is below the level of immunoassay detection.

These assay methods can comprise any immunoassay known in the art. Insome embodiments, the assay is performed in a liquid phase. In otherembodiments, the assay is performed on a solid phase, e.g., on a bead ora microplate, for example a 96 well microtiter plate. Nonlimitingexamples of immunoassays useful in these methods are a radioimmunoassay(see, e.g., U.S. Pat. No. 4,081,525), a Luminex® assay (see, e.g., Wonget al., 2008), a microarray assay, a fluorescence polarizationimmunoassay (see, e.g., U.S. Pat. No. 4,585,862), an immunoassaycomprising a Förster resonance energy transfer (FRET) signaling system(see, e.g., Blomberg et al., 1999; Mayilo et al., 2009), and enzymeimmunoassay (a.k.a. enzyme linked immunosorbent assay [ELISA]). As iswell known in the art, in ELISA, an enzyme combined with a substratethat becomes colored upon reaction with the enzyme provides the signalto quantify the antigen in the sample. See, e.g., O'Beirne and Cooper,1979.

Table 1 provides a summary of various immunoassays that can be utilizedfor detection of the hydroxycholesterols to which antibodies areprovided herewith. The general features of these assays are known in theart.

TABLE 1 24-OH cholesterol Detection Method probe Antibody Othercomponents Scintillation proximity Radio-iodinated ImmobilizedScintillant-coated plate or bead assay Fluorescence polarizationFluorescently-labeled Unlabeled assay Homogeneous time- Allophyocyanin-Europium resolved labeled cryptate-labeled fluorescence assay Amplifiedluminescence Biotin-labeled Acceptor bead- Streptavidin-labeled donorbead assay (ALPHAScreen) labeled Enzyme complementation Inactive enzymeImmobilized Enzyme acceptor, enzyme substrate assay donor-labeled (e.g.fluorogenic β-galactosidase substrate) ElectrochemilurainescenceRuthenium-labeled Immobilized Carbon electrode plates, chemical assaysubstrate and electrical stimulation

Any ELISA known in the art as useful for hapten detection can beutilized for the instant assays. ELISA for haptens generally utilize acompetitive format, i.e., where the hapten (here, 24HC) in the samplecompetes with a labeled hapten (e.g., a biotin-hapten or enzyme-haptenconjugate) for anti-hapten antibody binding sites such that less labeledhapten is bound when there is more hapten in the sample. Thus, in thesecompetitive assays, an increasing amount of hapten in the sample resultsin less enzyme bound to the solid phase, and consequently less coloredsignal. In such competitive assays, as defined herein, the sample can beadded with the labeled hapten to compete directly for antibody bindingsites, or the sample and labeled hapten can be added sequentially suchthat the labeled hapten simply binds where the sample hapten is notbound.

In some embodiments, the ELISA is a direct competitive ELISA, definedherein as where the 24HC hapten is directly bound to the signalingenzyme, or an indirect competitive ELISA, where the enzyme is bound toanother molecule, e.g., a second antibody, or streptavidin.

The ELISA assays provided herein can take any format known in the art.In some embodiments, a 24HC conjugate (e.g., 24HC-BSA or24HC-polylysine) is bound to the solid phase. In these assays, the 24HCantibody is added with the sample. Here, the 24HC in the sample competeswith the solid phase-bound 24HC conjugate for antibody binding sitessuch that less antibody binds to the solid phase when there is more 24HCin the sample. After washing, the amount of 24HC antibody bound to thesolid phase is measured, e.g., by utilizing in the competitive step a24HC antibody-enzyme conjugate, or by adding a second antibody-enzymeconjugate that binds to the bound 24HC antibody. A myriad of particularassays for 24HC with this configuration can be devised without undueexperimentation.

In other embodiments, the 24HC antibodies are bound to the solid phase,either directly or indirectly, the latter being where the solid phase iscoated with an anti-antibody (for example goat antibodies that bind torabbit IgG antibodies [goat anti-rabbit IgG]) and the 24HC antibodiesare bound to the anti-antibody. The anti-antibodies are also known as“second antibodies”. In these assays, the sample and a labeled hapten isadded to the solid phase to compete with antibody binding sites on thecoated solid phase. After washing, the signal is generated, whichmeasures the amount of labeled hapten that is bound to the solid phase.Numerous particular assays for 24HC with this configuration can bedevised without undue experimentation.

An illustration of the latter assay, where the 24HC antibodies are boundto the solid phase, is provided in the Example below. That assaycomprises:

a. noncovalently binding the second antibodies to the solid phase;

b. adding the 24S-hydroxycholesterol antibodies to the solid phase underconditions such that the second antibodies specifically bind to the24S-hydroxycholesterol antibodies;

c. adding the sample and a biotinylated 24S-hydroxycholesterol to thesolid phase under conditions such that the biotinylated24S-hydroxycholesterol binds to binding sites on the24S-hydroxycholesterol antibodies where 24S-hydroxycholesterol in thesample do not bind;

d. adding an avidin-enzyme conjugate to the solid phase under conditionssuch that the avidin-enzyme conjugate specifically binds to thebiotinylated 24S-hydroxycholesterol that bound in step c.;

e. adding a substrate to the enzyme in the avidin-enzyme conjugate,wherein the substrate is converted to a colored product that absorbslight at a specified wavelength in proportion to the amount of theenzyme bound to the solid phase;

f. measuring the absorbance of light at the specified wavelength,wherein the absorbance is inversely proportional to the amount of24S-hydroxycholesterol present in the sample.

With any of the above-described immunoassays, 24HC in the sample isquantified by comparing the signal generated (e.g., fluorescence in theFRET and fluorescence polarization assays, radioactivity inradioimmunoassays, absorbance in ELISAs) in the assay where the sampleis added to the signal generated in assays where a known amount of 24HCis added, i.e., comparing the sample signal to a standard curve.

The present invention is also directed to a kit for detecting andquantifying 24HC. The kit comprises 24HC antibodies. In someembodiments, the kit further comprises a protein conjugate or a biotinconjugate of 24S-hydroxycholesterol. In other embodiments, the kitfurther comprises a solid phase, e.g., beads or a microtiter plate. Inthese embodiments, the solid phase comprises a noncovalently boundprotein, where the bound protein is an antibody or a protein conjugateof 24S-hydroxycholesterol. In various embodiments, the kit furthercomprises an enzyme. Examples of such enzymes are alkaline phosphataseor horseradish peroxidase, covalently bound to 24HC or an antibody(e.g., a 24HC antibody or a second antibody). In additional embodiments,the kit further comprises 24HC, for example for use in a standard curve.

As discussed in the Background section of this application, 24HC is asubstrate for various cytosolic sulfotransferases (SULT) andUDP-glucuronosyltransferases (UGT). Sulfation involves the transfer ofthe sulfonate group of the obligate sulfonate donor3′-phosphoadenosine-5′-phosphosulfate (PAPS) to an acceptor compound,usually possessing a hydroxyl group, to form a sulfate ester. Typically,[³⁵S]PAPS is employed in the SULT assay. The assay is often terminatedby chloroform extraction and the sulfated products in the aqueous phaseare resolved by thin layer chromatography (TLC) on silica gel TLC platesand detected by autoradiography. However, to define the sites of sulfateconjugation, an LC-MS/MS approach is usually required. Nonradioactivereactions can be run in parallel with reactions containing [³S]PAPS andmonitored by TLC. The non-radioactive reactions are extracted withchloroform and the water phase loaded onto a Sep-Pak cartridge, washedwith water and eluted with MeOH. The eluates can be concentrated byevaporation under a stream of N₂. Identification of the sulfatedcompounds is by HPLC-mass spectroscopy using, for example, a SciexAPI-4000 Triple Quadrupole mass spectrometer with two Perkin Elmerseries 200 micropumps (Perkin Elmer Life and Analytical Sciences,Boston, Mass.) and a Synergi Fusion (100×2 mm ID) analytical C-18 columnwith a C-18 guard column (Phenomenex).

In an analogous manner, the glucuronidation reaction can be monitored asthe transfer of the glucuronosyl group from UDP[U-¹⁴C]glucuronic acid tothe target substrate. HPLC on a polar amino-cyano bonded phase column isemployed to separate radioactive glucuronides from unmetabolizedUDP[U-14C]glucuronic acid (Coughtrie et al., 1986).

The immunoassays described herein can be utilized to detect activity ofUGT and SULT or any other enzymes that alter a hydroxycholesterol suchthat it no longer binds to the antibodies described herein. For example,the extracerebral double conjugation of 24-OH cholesterol to generate(24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide can be analyzed byimmunoassay using the immunoassays of the present invention incombination with, e.g., immunoassays selective for sulfation, oralternatively in combination with techniques allowing the selectiveseparation of the sulfated form of the analyte from the nonsulfatedform. For example, approaches that allow the selective precipitation ofthe sulfated form of the analyte, as described in U.S. patentapplication Ser. No. 12/806,950, filed Aug. 24, 2010, would facilitatethe analysis. Such an approach eliminates the need for thetime-consuming analysis of UGT and SULT activity now utilized, asdescribed above. The potential for high throughput analysis as can beemployed with the immunoassays described herein, as well as the highsensitivity of the immunoassays, provides opportunities to utilize theseimmunoassays to monitor phase II drug metabolism assays and screen drugsand new chemical entities for their capacity to modulate phase II enzymeactivity in native or recombinant fractions.

Thus, the present invention is also directed to a method of detecting anenzyme or enzymes utilized in phase II drug metabolism in a sample,where the enzyme or enzymes are capable of altering22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol. The method comprisescombining the sample with 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterolfor a time sufficient for the enzyme or enzymes to modify the22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol, then assaying the sampleusing any of the above-described immunoassays. In these methods, areduction in the amount of the 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol detected by the assay indicates that the samplecomprises the enzyme or enzymes.

In some embodiments of these methods, the enzyme is a cytosolicsulfotransferase, a UDP-glucuronosyltransferase, or a combinationthereof. In certain specific embodiments, the enzyme or enzymes arecapable of altering 24S-hydroxycholesterol.

These methods can also be utilized to determine whether a compound iscapable of modulating the enzyme or enzymes, or reducing theirexpression (the latter case where the sample includes cells that producethe enzyme or enzymes). In these embodiments, the compound is added tothe sample with the enzyme or enzymes. If the compound modulates theenzyme or enzymes, the difference in the amount of the22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol detected by theimmunoassay when the compound is included vs. when the compound isexcluded indicates that the compound modulates the enzyme. For example,if the compound inhibited or reduced the expression of the enzyme orenzymes, less of the hydroxycholesterol would be altered, and theimmunoassay would show more of the hydroxycholesterol present than whenthe compound was not included with the enzyme or enzymes. Conversely, ifthe compound enhanced the activity, or increased expression of theenzyme or enzymes, more of the hydroxycholesterol would be altered, andthe immunoassay would show less of the hydroxycholesterol present thanwhen the compound was not included with the enzyme or enzymes.

In a specific embodiment, inhibitor screening for UGT and SULT can beaccomplished using a competitive binding assay format. A decrease in theformation of 24-OH cholesterol metabolite compared to the vehiclecontrol is used to calculate an IC50 value of the test agent. Forexample, a UGT isozyme inhibition assay can be performed as follows: TheUGT substrate, 24-OH cholesterol, is incubated with a cDNA-expressedhuman UGT isoform, such as UOT1A1, as well as with UDP-glucuronic acid(UDPGA), and a range of test compound concentrations (typically 0.4-100μM) for 30 min at 37° C. Optionally, a microsomal preparation can alsobe incorporated to enhance enzyme activity. Due to the luminal locationof the UGT's within the endoplasmic reticulum of microsomalpreparations, the passage of the water soluble cofactor UDPGA to theactive site can be challenging. In order to circumvent this latencyphenomenon, the pore forming agent, alamethicin, can be included toimprove access to the active site. At the end of the incubation period,the formation of the metabolite, 24-OH cholesterol 3-glucuronide ismonitored by immunoassay, using antibodies of the present invention, ateach of the test compound concentrations. A decrease in the formation ofthe metabolite compared to vehicle control is used to calculate an IC50value of the inhibitor (test compound concentration which produces 50%inhibition of UGT1A1). The selective UGT1A1 inhibitor, silybin, can beused as the positive control inhibitor in the described assay.

These methods can be further utilized to quantify the enzyme or enzymes,e.g., by comparing the reduction in the amount of the22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol detected by the assay inthe sample with the reduction in the amount of 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol present when treated by a standard amount of theenzyme or enzymes.

The above-described immunoassays can also be utilized in a method todetect an enzyme that synthesizes 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol in a sample. The method comprises combining thesample with cholesterol for a time sufficient for the enzyme to modifythe cholesterol, then assaying the sample using any of theabove-described immunoassays. In these methods, an increase in theamount of 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesteroldetected by the assay indicates that the sample comprises the enzyme. Insome embodiments of these methods, the enzyme is CYP46 and the assaydetects 24S-hydroxycholesterol.

In certain embodiments of these methods, the enzyme in the sample isquantified by comparing the increase in the amount22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol or 27-hydroxycholesterol detected by the assay inthe sample with the increase in the amount of 22-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or27-hydroxycholesterol present when cholesterol is treated by a standardamount of the enzyme.

Since reductions in plasma 24HC levels are associated with certaindiseases, in particular multiple sclerosis and Huntington's disease (seeBackground above), the immunoassays described herein can be used tomonitor the progression and efficacy of treatment for those diseases.

Thus, in some embodiments, a method of evaluating progression ofmultiple sclerosis in a patient is provided. The method comprisesmeasuring 24S-hydroxycholesterol in blood plasma or serum of the patientover time by the above-described assay method. In this method,progression of multiple sclerosis is indicated by decreasing levels of24S-hydroxycholesterol in the blood plasma or serum over time.

Also provided is a method of determining whether a treatment formultiple sclerosis in a patient is effective is also provided. Themethod comprises measuring 24S-hydroxycholesterol in blood plasma orserum of the patient over time by the above-described assay method. Inthis method, the treatment is effective if the 24S-hydroxycholesterollevels do not decrease over time.

A method of evaluating progression of Huntington's disease in a patientis also provided. The method comprises measuring 24S-hydroxycholesterolin blood plasma or serum of the patient over time by the above-describedassay method. In this method, progression of Huntington's disease isindicated by decreasing levels of 24S-hydroxycholesterol in the bloodplasma or serum over time.

Additionally provided is a method of determining whether a treatment forHuntington's disease in a patient is effective is additionally provided.The method comprises measuring 24S-hydroxycholesterol in blood plasma orserum of the patient over time by the above-described assay method. Inthis method, the treatment is effective if the 24S-hydroxycholesterollevels do not decrease over time.

Preferred embodiments are described in the following example. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example Preparation of Antisera to 24S-hydroxycholesterol and its Use inImmunoassay Detection of Same

Since 24S-hydroxycholesterol (24HC) is a hapten that is too small toillicit an antibody immune response by itself, it must be covalentlycoupled to a carrier protein, which, when injected in a vertebrate,presents 24HC to the vertebrate's immune system as an epitope on theprotein, such that antibodies are generated to the 24HC epitope.However, 24HC does not have a moiety to which it could be convenientlyconjugated to a carrier protein. Therefore, the 3-O-succinoyl derivativeof 24HC was prepared as follows:

Preparation of 3-O-Succinoyl-24S-hydroxycholesterol

To 24HC (21 mg [0.052 mmol]) in 3 mL dry pyridine was added succinicanhydride (10 mg [0.10 mmol]). The mixture was heated under Ar at 80° C.for 3 days. The reaction was then diluted with ethyl acetate and washedwith 1M HCl and brine. Flash chromatography (2% MeOH/CH2Cl2) yielded 12mg (46%) of product. This scheme is illustrated below:

An activated ester of 3-O-succinoyl-24S-hydroxycholesterol,3-O-succinoyl-24S-hydroxycholesterol N-hydroxysuccinimide ester, wasthen prepared as follows:

Preparation of 3-O-Succinoyl-24(S)-hydroxycholesterolN-hydroxysuccinimide ester

To 3-O-Succinoyl-24(S)-hydroxycholesterol (9.2 mg [0.018 mmol]) andN-hydroxysuccinimide (NHS) (2.3 g [20 mmol]) in 2 mL dry THF was added1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (3.9 mg [0.020 mmol]). Thereaction mixture was stirred overnight at room temperature. It was thendiluted with diethyl ether and washed with water and brine. Flashchromatography (100% CH₂Cl₂) gave 4.7 mg (43%) of the desired product.This scheme is illustrated below:

The 3-O-Succinoyl-24S-hydroxycholesterol NHS ester was then conjugatedto the carrier protein keyhole limpet hemocyanin (KLH) as follows:

Preparation of 3-O-Succinoyl-24S-hydroxycholesterol-KLH

1. Ten mg of KLH lyophilized in 0.1 M sodium phosphate, 0.15 M NaCl; pH7.2 (Pierce product #77600) was resuspended in 1 mL water.

2. Four mg of the 3-O-succinoyl-24S-hydroxycholesterol NHS ester wasresuspended in 100 μL of dimethyl sulfoxide.

3. The 3-O-succinoyl-24S-hydroxycholesterol NHS ester solution was addeddropwise to the KLH solution 10 μL at a time. After 70 μL was added, aprecipitate was observed and 0.5 mL of H₂O was added to clear thesolution before adding the remainder of the NHS ester.

4. The solution was gently mixed on a tilting platform mixer for 1 hourat room temperature and then transferred to a dialysis bag (1 kD poresize). The solution was dialysed for 4.5 hours at room temperatureagainst 1 liter 0.1 M sodium phosphate, 0.15 M NaCl; pH 7.2. Thedialysis buffer was changed and dialysis continued overnight. Thedialysis buffer was changed one more time and dialysis was completedafter an additional 6.5 hours.

5. The final solution was brought to 10 mL final volume and aliquoted.

This 3-O-Succinoyl-24S-hydroxycholesterol-KLH conjugate was used as animmunogen to elicit antibodies in rabbits by the following protocol:

Immunization Protocol

1. Female New Zealand White rabbits were immunized intradermally with500 μg of 24S-hydroxycholesterol-KLH conjugate in PBS. The immunogenwith complete adjuvant was prepared by mixing 0.5 mL of 1 mg/mLhydroxycholesterol-KLH conjugate with 0.5 mL Freund's complete adjuvantand emulsifying by passage through glass syringes.

2. Prebleeds were collected 1 day prior to the first immunization.

3. Rabbits were injected 1 mL of immunogen intradermally over 10 siteson day 1.

4. Immunogen with incomplete adjuvant was prepared by mixing 0.5 mL of 1mg/mL 24S-hydroxycholesterol-KLH conjugate with 0.5 mL Freund'sincomplete adjuvant and emulsifying by passage through glass syringes.

5. Rabbits were injected with 1 mL of this immunogen intradermally over10 sites on day 21, 42 and 63.

6. Rabbits were bled on Day 74 and Day 77.

7. Rabbits were injected with 1 mL of immunogen with incomplete adjuvant(described in 4. above) intradermally over 10 sites on day 84.

8. Rabbits were bled on Day 95.

11. Rabbits were injected with 1 mL of immunogen with incompleteadjuvant intradermally over 10 sites on day 105.

12. Rabbits were bled on Day 116.

14. Rabbits were injected with 1 mL of immunogen with incompleteadjuvant intradermally over 10 sites on day 126.

15. Rabbits were bled on Day 136 and 138.

In some cases, the antisera was screened in an ELISA using 96 wellmicrotiter plates coated with a 3-O-succinoyl-24S-hydroxycholesterollinked to bovine serum albumin (BSA), prepared by conjugating the3-O-succinoyl-24S-hydroxycholesterol NHS ester described above to BSA bythe method described above with KLH. This screening protocol is asfollows:

Antisera Screening Assay Using 24S-hydroxycholesterol-BSA

1. 100 μL of 24(S)Hydroxycholesterol-BSA conjugate was coated to plateat a concentration of 1 μg/mL in 10 mM sodium phosphate buffer pH 8.0overnight at 4° C. Plates were blocked for 1 hour in 10 mM sodiumphosphate, 150 mM sodium chloride, 1% BSA buffer.

2. Rabbit antisera was serially diluted in 100 mM phosphate, 150 mMNaCl, pH 7.2 assay buffer and 100 μL of each dilution was transferred toindividual wells on a 96 well plate. The plate was incubated at roomtemperature for 1 h with orbital shaking.

3. The plates were washed 4 times with 300 μL of 50 mM Tris, 100 mMNaCl, 0.05% Tween-20 buffer.

4. Goat anti-rabbit-horseradish peroxidase (HRP) conjugate was dilutedto 200 ng/mL and 100 μL was added to each well and incubated for 1 hourat room temperature.

5. The plate was washed 4 times with 300 μL of 50 mM Tris, 100 mM NaCl,0.05% Tween-20 buffer to remove excess HRP conjugate. 100 μL of TMBsubstrate solution was added to each well. The plates were incubated atroom temperature for 30 min. An HRP-catalyzed reaction generates a bluecolor in the solution.

6. After color development, 50 μL of 1N HCl stop solution is added toeach well to stop the substrate reaction. The HCl solution converts theblue color to yellow. The resulting yellow color was read at 450 nm. Theintensity of yellow color (read as absorbance at 450 nm) is directlyproportional to the amount of anti-24S-hydroxycholesterol antibody inthe in the sample.

In other cases, the antisera were screened using an assay that employs abiotinylated 3-O-succinoyl-24(S)-hydroxycholesterol, which was preparedas follows:

Preparation of biotinylated 3-O-succinoyl-24S-hydroxycholesterol

To 3-O-Succinoyl-24S-hydroxycholesterol N-hydroxysuccinimide ester (4.7mg [7.8 μmol]) in 1 mL dry CH₂Cl₂ was addedbiotinyl-3,6,9-trioxaundecanediamine (3.3 mg [7.8 μmol]). After stirringovernight at room temperature, the mixture was subjected to flashchromatography (10% MeOH/CH₂Cl₂) to yield 4.4 mg (62%) of product. Thisscheme is illustrated below:

The screening assay using the biotinylated3-O-succinoyl-24S-hydroxycholesterol reagent was as follows:

Antisera Screening Assay Using Biotinylated 24HC

1. A microtiter plate (96 well) was coated with goat anti-rabbit IgGantibody. Rabbit antiserum to 24HC (100 μl), serially diluted in 100 mMphosphate, 150 mM NaCl, pH 7.2 assay buffer, was added to 100 μLbiotinylated 24HC diluted in the same buffer before adding to themicrotiter plate wells coated with a goat anti-rabbit IgG antibody. Theplate was incubated at room temperature for 1 hour with orbital shaking.During this incubation the antibody binds the biotinylated24S-hydroxycholesterol and becomes bound to the goat anti-Rabbit IgGcoated on the plate surface.

2. The plate is washed 4 times with 300 μL of 50 mM Tris, 100 mM NaCl,0.05% Tween-20 buffer. After washing only bound 24S-hydroxycholesterolor bound biotinylated 24S-hydroxycholesterol remains.

3. Streptavidin conjugated to horseradish peroxidase was diluted toworking concentration in 100 mM phosphate, 150 mM NaCl, pH 7.2 assaybuffer and 200 μL was added to each well to allow detection of the boundbiotinylated 24S-hydroxycholesterol. The plate was incubated at roomtemperature for 30 minutes.

4. The plate was washed 4 times with 300 μL of 50 mM Tris, 100 mM NaCl,0.05% Tween-20 buffer to remove excess HRP conjugate. TMB substratesolution (200 μL) is added to each well. The plates were incubated atroom temperature for 30 min.

5. After color development, 50 μL of 1N HCl stop solution is added toeach well to stop the substrate reaction. The resulting yellow color wasread at 450 nm. The intensity of yellow color is directly proportionalto the amount of anti-24S-hydroxycholesterol antibody in the in thesample.

The following protocol was used to detect varying amounts of 24HC tocreate a standard curve for 24HC:

24HC Enzyme Immunoassay Protocol

1. Known concentrations of 24HC were prepared in 100 mM phosphate, 150mM NaCl, 1% BSA, pH 7.2 assay buffer containing biotinylated 24HS(described above) and added to wells of a 96 well microtiter plate thatwere coated with a goat anti-rabbit IgG antibody. Rabbit polyclonalantibody to 24HC was diluted to a limiting concentration in 100 mM Tris,150 mM NaCl, 1% BSA buffer and 100 μL was added to each well. The platewas incubated at room temperature for 1 hour with orbital shaking.During this incubation the anti-24HC antibody binds to the goatanti-rabbit IgG antibody on the plate. Also, the 24HC standard competeswith the biotinylated 24HC for anti-24HC antibody binding sites. Thus,with more 24HC standard, less biotinylated 24HC binds to the antibodybinding sites on the plate.

2. Following this incubation, the plate was washed 4 times with 300 μLof 50 mM Tris, 100 mM NaCl, 0.05% Tween-20 buffer. After washing, bound24HC or bound biotinylated 24HC is bound to the binding sites on theanti-24HC antibodies.

3. Streptavidin conjugated to HRP (200 μL), diluted in 100 mM phosphate,150 mM NaCl, 1% BSA, pH 7.2 assay buffer, was then added to each well toallow detection of the biotinylated 24HC. The plate was then incubatedat room temperature for 30 minutes.

4. The plate was then washed 4 times with 300 μL of 50 mM Tris, 100 mMNaCl, 0.05% Tween-20 buffer to remove excess streptavidin-HRP conjugate.TMB substrate solution (200 μL) is added to each well. The plates wereincubated at room temperature for 30 min.

5. After color development, 50 μL of 1N HCl stop solution is added toeach well to stop the substrate reaction. The resulting yellow color wasread at 450 nm. The resulting yellow color was read at 450 nm. Theintensity of yellow color is inversely proportional to the concentrationof 24HC that was added to the well.

A typical standard curve for this assay is shown in FIG. 1. The multipleregression line shown therein has an R² of 0.996. The detectable rangefor 24HC for this assay is as shown is 1.56-100 ng/mL. The total assaytime is 2 h.

Cross-reactivity of the antisera tested above (Day 77 bleed, rabbit #72)to the following structurally similar molecules was determined:

Results of that cross-reactivity study are shown in Table 2:

TABLE 2 Molecule % Cross reactivity Cholesterol <0.00122-Hydroxycholesterol 0.15 DHEA 0.05This study shows that the cross-reactivity to these structurally similarcompounds is minimal.

Cross-reactivities of antisera taken from another rabbit at Day 77(rabbit #71), subjected to the same immunization protocol, as well as arepeat of the rabbit #72 antisera, were also determined. The results areshown in Table 3.

TABLE 3 Bleed #71 Bleed #72 Molecule % Cross reactivity % Crossreactivity Cholesterol 0.002 0.003 22-Hydroxycholesterol 0.020 0.1125-Hydroxycholesterol 0.19 0.725 27-Hydroxycholesterol 0.022 <0.001 DHEA0.015 0.018As with the assay described in Table 2, the cross-reactivities ofstructurally similar compounds with these two antisera were minimal. Thesmall differences in cross-reactivities described in Table 1 and the“Bleed #72” data in Table 3 represent variation in the assay.

Based on the studies described in this Example, the antisera to 24HC isuseful for quantifying 24HC in, e.g., plasma, cerebrospinal fluid, andtissue.

REFERENCES

-   Alexandrov at al., 2005, Neuroreport. 21:909-13.-   Axelson, 1991, J. Lipid Res. 32:1441-8.-   Björkhem et al., 2009, Mol. Aspects Med. 30:171-9.-   Birö at al., 2007, J. Lipid Res. 48:19-29.-   Blomberg et al., 1999, Clin. Chem. 45:855-61.-   Bretillon et al., 2000, J. Lipid Res. 41:840-5.-   Bryleva et al., 2010, Proc. Natl. Acad. Sci. USA 107:3081-6.-   Burkard at al., 2004, J. Lipid Res. 45:776-81.-   Charlton and Porter, 2002, Meth. Mol. Biol. 178:159-71.-   Cook et al., 2009, Drug Metab Dispos. 37:2069-78.-   Coughtrie et al., 1986, Anal. Biochem. 59:198-205.-   DeBarber et al., 2008, Anal. Biochem. 381:151-3.-   Dijkstra et al., 1996, J. Immunol. 157:2006-13.-   Garcia et al., 2009, J. Mol. Neurosci. 39:342-5.-   Hudry et al., 2010, Mol. Ther. 18:44-53.-   Irizarry, 2004, NeuroRx 1:226-34.-   Jennings et al., 1999, Am. J. Physiol. 277:G-1017-26.-   Kölsch et al., 2004, Neurosci. Lett. 368:303-8.-   Koschack et al., 2009, Neurobiol. Aging 30:898-902.-   Leoni, 2009, Scand. J. Clin. Lab Invest. 69:22-5.-   Leoni et al., 2008, Brain 131:2851-9.-   Leoni et al., 2010, Biochem. Soc. Trans. 38:1021-5.-   Lütjohann and von Bergmann, 2003, Pharmacopsychiatry 36 Suppl.    2:S102-6.-   Lütjohann et al., 1996, Proc. Natl. Acad. Sci. USA 93:9799-804.-   Lütjohann et al., 2000, J. Lipid Res. 41:195-8.-   Masterman et al., 2002, Neurosci. Lett. 331:163-6.-   Mayilo et al., 2009, Analytica Chimica Acta 646:119-22.-   Mcintosh et al, 2008 Chapter 1. Fluorescent Sterols for the Study of    Cholesterol Trafficking in Living Cells, pp. 1-20 In: Probes and    Tags to Study Biomolecular Functions: For Proteins, RNA and    Membranes, Lawrence W. Miller (Editor), Wiley Press.-   O'Beirne and Cooper, 1979, J. Histochem. Cytochem. 27:1148-62.-   Papassotiropoulos et al., 2002, J. Psychiatr. Res. 36:27-32.-   piercenet.com/browse.cfm?fldID=4E018AA6-5056-8A76-4E57-3BC84C88A328.-   Sato et al., 1976, Biomedicine. 15:385-9.-   Shafaati et al., 2007, Neurosci. Lett. 425:78-82.-   Solomon et al., 2009, Neurosci. Lett. 462:89-93.-   Sparrow et al., 1999, J. Lipid Res. 40:1747-57/-   Teunissen et al., 2003, Neurosci. Lett. 347:159-62.-   Teunissen et al., 2005, Lancet Neurol. 4:32-41.-   Valenza et al., 2007, Hum. Mol. Genet. 16:2187-98.-   Vega and Weiner, 2007, J. Mol. Neurosci. 33:51-5.-   Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.-   Wong et al., 2008, Cancer Epidemiol. Biomarkers 17:3450-6.-   Yinsong et al., 2007, Carbohydrate Polymers 69:597-606.-   Zhao et al., 2009, Neurosci. 164:398-403.-   U.S. Pat. No. 4,081,525.-   U.S. Pat. No. 4,585,862.-   U.S. patent application Ser. No. 12/806,950.

In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

What is claimed:
 1. A method of making antibodies that specifically bindto hydroxycholesterol, 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol,the method comprising a. preparing an immunogen by conjugating ahydroxycholesterol selected from hydroxycholesterol,22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol,26-hydroxycholesterol and 27-hydroxycholesterol or a derivative ofhydroxycholesterol, 22-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 26-hydroxycholesterol and 27-hydroxycholesterolto a carrier protein; b. immunizing a vertebrate with the immunogenunder conditions such that the immune system of the vertebrate makesantibodies to the hydroxycholesterol; and c. collecting the antibodiesto the hydroxycholesterol from the vertebrate.
 2. The method of claim 1,wherein the hydroxycholesterol is an O-succinoyl derivative.
 3. Themethod of claim 2, wherein the hydroxycholesterol derivative is3-O-succinoyl-24S-hydroxycholesterol


4. The method of claim 1, wherein the carrier protein is keyhole limpethemocyanin, bovine serum albumin or ovalbumin.
 5. The method of claim 1,wherein the vertebrate is a rabbit, a goat, a mouse, a chicken, or asheep.
 6. The method of claim 1, wherein the vertebrate is a rabbit. 7.The method of claim 1, wherein the vertebrate is a mouse.
 8. The methodof claim 1, wherein the antibodies collected from the vertebrate arepolyclonal antibodies.
 9. The method of claim 1, wherein the antibodiescollected from the vertebrate are monoclonal antibodies.